Sexual dimorphism is a fundamental feature of virtually all animals, affecting many tissues and cell types in the body. In the nematode Caenorhabditis elegans, the activity of a single gene, tra-1, is necessary and sufficient to dictate whether the entire body develops as male or female. If tra-1 is active, all tissues differentiate as female, whereas if tra-1 is inactive, all tissues differentiate as male. Thus tra-1 is an ideal candidate for study of sexual differentiation. In addition, C. elegans is an ideal organism for such studies because the two sexes differ extensively, development can be analyzed at the single cell level, and powerful molecular and genetic tools exist. In this project I want to answer two basic questions: first, what controls the activity of tra-1, and second, when tra-1 is active, what genes does it, in turn, control to cause female sexual differentiation. Elegant genetic studies have shown that a series of negative regulators converts the X chromosome to autosome ratio of the embryo into an 'on' or 'off' state of tra-1. How is this accomplished? Molecular and genetic studies of tra-1 revealed that control of its activity requires a short domain of the TRA-1 protein. To identify regulators that bind this domain and inactivate TRA-1, we are performing a yeast two-hybrid screen. We will identify proteins that bind to the critical domain of TRA-1 and test their function in vivo. Using this portion of TRA-1 as a 'bait', we have already identified several promising candidate regulators which we propose to study further. These experiments will complete the link between upstream regulators and tra-1. How does tra-1 direct sexual differentiation? To find out, I propose to study genes that are regulated by tra-1, using two approaches. The first is to study mab-3, a gene whose properties (a sexual regulator acting downstream of tra-1) suggest it is a tra-1 target. mab-3 has been localized to a short chromosomal interval by transformation rescue, and will be cloned. mab-3 will be tested for transcriptional regulation by tra-1, and its role in regulating yolk protein expression in the intestine will be studied. The second approach is to identify new genes that are controlled by tra-1, using a representational difference analysis (RDA) PCR method. I will identify sex-specific mRNAs from the two sexes. From this screen, I will select a small number of genes for further study, concentrating on those that appear to be directly regulated by tra-1. These experiments will ultimately reveal in molecular detail how tra-1 controls sexual differentiation.